Nitric oxide (NO) increases cytosolic guanylate cyclase activity and thereby activates the cGMP signal transduction pathway. The cAMP and Ca2+/phospholipid signal transduction pathways activate transcription factors that bind to the cAMP response element (CRE) and phorbol ester response element (TRE), respectively. Little is known about transcriptional regulation of gene expression by NO/cGMP. In transient and stable transfection experiments and in microinjection studies we found that three different NO-releasing agents and two membrane-permeable cGMP analogs activated TRE-regulated but not CRE-regulated reporter genes in rodent fibroblast and epithelial cell lines. Activation of TRE-regulated genes by NO-releasing agents and cGMP analogs appeared to be mediated by the AP-1 (Jun/Fos) transcription factor complex because we observed increased DNA binding of AP-1 and increased junB and c-fos mRNA in cells treated with these agents. The mechanism of gene activation by NO/cGMP was distinct from that used by phorbol esters and cAMP because it was not associated with c-jun mRNA induction and was not observed with CRE-containing promoters.

The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.

Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3,5-cyclic monophosphate and 8-bromoguanosine-3,5-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase I␤ expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor

The human immunodeficiency virus (HIV-1) differentially controls viral protein expression at the level of splicing as well as nuclear export of incompletely spliced viral RNA. This process, mediated by the Rev protein, interfaces with cellular components involved in post-transcriptional gene regulation. While a number of reviews have focused on the host proteins (i.e., Crm1, importin-beta and nucleoporins) that specifically regulate shuttling of Rev between the nucleus and cytoplasm, we could find no systematic review of other cellular proteins implicated in Rev function. Therefore, we will here focus on other Rev cofactors (eIF5a, hRIP, Sam68, RNA helicases, etc) and the role they play in Rev/RRE function and HIV-1 replication.

During hexamethylene bisactamide (HMBA)-induced dierentiation of murine erythroleukemia (MEL) cells erythroid genes are transcriptionally activated while cmyb and several other nuclear proto-oncogenes are down-regulated. Dierentiation is inhibited by cAMP analogues and the adenyl cyclase-stimulating agent forskolin. We found that these drugs prevented the late down-regulation of c-myb which is known to be critical for MEL cell dierentiation. Since the increase in c-myb mRNA levels was due to increased mRNA transcription, we examined the transcription factors NF-kB and AP-1 which have been implicated in the regulation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-kB recognition sequence in c-myb intron I was strongly induced by 8-Br-cAMP or forskolin; induction was delayed and correlated with the up-regulation of cmyb. The cAMP-induced NF-kB complex contained p50 and RelB; in co-transfection assays, p50 and RelB transactivated a reporter construct containing the c-myb intronic NF-kB site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increased the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells which could cooperate with NF-kB. We conclude that inhibition of MEL cell dierentiation by pharmacological doses of cAMP can be explained by the up-regulation of c-myb which is mediated, at least in part, by NF-kB p50/RelB heterodimers.

Sam68 functionally complements for, as well as synergizes with, HIV-1 Rev in Rev response element (RRE)-mediated gene expression and virus production. Furthermore, C-terminal deletion/point mutants of Sam68 (Sam68ΔC/Sam68-P21) exert a transdominant negative phenotype for Rev function and HIV-1 production. However, the relevance of Sam68 in Rev/RRE function is not well defined. To gain more insight into the mechanism of Sam68 in Rev function, we used an RNAi (RNA interference) strategy to create stable Sam68 knockdown HeLa (SSKH) cells. In SSKH cells, Rev failed to activate both RRE-mediated reporter gene [chloramphenicol acetyltransferase (CAT) and/or gag] expressions. Importantly, reduction of Sam68 expression led to a dramatic inhibition of HIV-1 production. Inhibition of the reporter gene expression and HIV production correlated with the failure to export RRE-containing CAT mRNA and unspliced viral mRNAs to the cytoplasm, confirming that SSKH cells are defective for Rev-mediated RNA export. Taken together, these results suggest that Sam68 is involved in Rev-mediated RNA export and is absolutely required for HIV production.

No abstract

Sam68 (Src-associated protein in mitosis 68 kDa) is a multifunctional protein, known to govern cellular signal transduction, transcription, RNA metabolism, proliferation, apoptosis and HIV-1 replication. Although intrinsic mechanisms that modulate Sam68 function are beginning to emerge, the regulatory events contributing to its expression remain elusive. We previously reported that heat shock protein-22 (Hsp22) antagonizes Sam68 function in rev-response element (RRE)-mediated gene expression. We now demonstrate that Sam68 levels correlate inversely with Hsp22 in a variety of cells, including U87, Jurkat, 293T and U-937. In U87 glioblastoma cells, which contained high levels of Hsp22 than other cell lines tested, Hsp22 knockdown dramatically increased both Sam68 mRNA and protein, altered cellular morphology and enhanced cell proliferation. This heightened proliferation was associated with a sharp decrease in G0/G1 and a corresponding increase in S and G2/M phases in exponentially growing cultures. The increased S phase population in turn correlated with enhanced expression of cell cycle regulatory proteins such as cyclin E, cyclin A, ribonucleotide reductase (RNR) and proliferating cell nuclear antigen (PCNA), which are required for the transition of cells from G1 to S phase. Collectively, our results demonstrate for the first time that Hsp22 regulates Sam68 expression and the ratio of Sam68 to Hsp22 may determine the proliferative potential of glioblastoma cells.